The natural world changes continually and the nervous system must recognize and adjust to this change for optimal behavior and survival. Norepinephrine (NE) from the locus coeruleus (LC) and its interactions with the prefrontal cortex (PFC) have been suggested to enable behavioral flexibility under conditions of uncertainty about outcomes. Much of the evidence for the role of LC in this context comes from measurements of pupil diameter, which have been suggested to correlate with the state of LC. However, there is no conclusive evidence linking single-cell LC activity to either pupil diameter or uncertainty, nor is there evidence directly linking LC-NE activity in PFC to uncertainty. The goal of this proposal is to bridge this gap by utilizing genetic and physiological manipulations in behaviorally-trained mice and implementing the following aims: Aim 1: Determine the relationship between uncertainty, LC activity, and pupil diameter; Aim 2: Determine the causal role of LC in enabling behavioral flexibility; Aim 3: Establish the relationship between NE-induced neuromodulation in PFC and behavioral flexibility. Aim 1 will utilize cell-type specific tools to unambiguously identfy and record from LC neurons in vivo in mice performing an uncertainty-based task, a goal which has not yet been realized for the NE neuromodulatory system. This will give clear evidence regarding the role of LC in behavioral flexibility during uncertainty and will clarify the use of ppil diameter as an index of LC physiology. Aim 2 will test causality and involve the use of optogenetic modulation of LC during this task to manipulate behavioral flexibility. Aim 3 will involve the use of two-photon imaging to characterize the activity of LC-NE axons projecting to PFC to investigate the role of LC- NE in PFC in enabling behavioral flexibility during uncertainty. Given that individuals suffering from any of a number of psychiatric disorders show deficits in recognizing and adjusting to change, defining the relevant neural circuitry in this context may be necessary to better understand and ultimately treat these disorders. These studies will not only aid in an understanding of the role of the NE system during uncertainty, but will also shed light on our abilities to learn and adapt in general.